The overall goal of this project is to elucidate the genetic pathways and cellular mechanism that mediate ventral folding morphogenesis. Human embryos undergo ventral folding morphogenesis during the fourth week of gestation. Despite the different topologies of the epiblast in early human and mouse embryos (a flat disc in the former, a hollow cylinder in the latter), a number of key aspects of ventral folding morphogenesis are conserved: internalization of the gut, formation of a linear heart tube, closure of the ventral body wall, and encasement of the fetus in the amniotic membrane. Abnormalities in ventral morphogenesis underlie a number of birth defects associated with incomplete body wall closure (gastroschisis, development of intestines outside the abdominal wall; ectopia cordis, location of heart outside the thoracic cavity); such aberrations of ventral folding occur in ~ 1/2000 live births. An absence of knowledge about the signaling pathways and cell populations directing ventral folding morphogenesis in mammals has precluded systematic study of the genetics and cell biology underlying this developmental process. The proposed studies build directly on our recent finding that the Anterior Visceral Endoderm (AVE), a well defined signaling center in the pre-streak (PS) and Early Streak (ES) mouse embryo, plays a central role in initiating and directing anterior ventral folding morphogenesis. Tissue-specific mouse mutant analyses have shown that the Bone Morphogenetic Protein (BMP) pathway is a key regulator of anterior ventral folding morphogenesis. BMP2 expressed by the AVE signals to epiblast derivatives during gastrulation to orchestrate the initial stages of ventra morphogenesis; including formation of the foregut invagination and placement of the heart caudal to the head. Our central hypothesis is that AVE-expressed BMP2 directs foregut invagination and head fold morphogenesis by signaling to one or more distinct epiblast-derived cell types: gut endoderm, anterior neuroectoderm, and/or cardiac mesoderm. Upon receipt of the AVE-derived BMP2 signal, target tissues undergo morphogenetic changes in cell behaviors that coordinate the generation of foregut invagination with a rostral-caudal shift in the positions of head and heart. Three specific aims will investigate this hypothesis. Aim 1 uses lineage-specific Cre transgenes and a conditional allele of Bmpr1a to determine if AVE-expressed Bmp2 signals to definitive endoderm, ectoderm and/or mesoderm to initiate ventral folding morphogenesis. Aim 2 investigates whether a VE specific null allele of Bmp2 genetically interacts with null alleles of Gata4 and Hgs/Hrs, mutants known to also disrupt ventral folding morphogenesis. Aim3 maps cis-acting elements that target transcription to the proximal AVE using Bmp2-LacZ BAC reporter transgenic mouse lines; this will allow us to develop expression vectors for in vivo studies on the functional properties of the BMP2-expressing population in the proximal AVE.